Subfamily-Specific Adaptations in the Structures of Two Penicillin-Binding Proteins from Mycobacterium tuberculosis

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis

Crystal structure of the C-terminal half of tropomodulin and structural basis of actin filament pointed-end capping.

Tropomodulin is the unique pointed-end capping protein of the actin-tropomyosin filament. By blocking elongation and depolymerization, tropomodulin regulates the architecture and the dynamics of the filament. Here we report the crystal structure at 1.45-A resolution of the C-terminal half of tropomodulin (C20), the actin-binding moiety of tropomodulin. C20 is a leucine-rich repeat domain, and this is the first actin-associated protein with a leucine-rich repeat. Binding assays suggested that C20 also interacts with the N-terminal fragment, M1-M2-M3, of nebulin. Based on the crystal structure, we propose a model for C20 docking to the actin subunit at the pointed end. Although speculative, the model is consistent with the idea that a tropomodulin molecule competes with an actin subunit for a pointed end. The model also suggests that interactions with tropomyosin, actin, and nebulin are all possible sources of influences on the dynamic properties of pointed-end capping by tropomodulin

Structure of a tropomyosin N-terminal fragment at 0.98 Å resolution

The crystal structure of the N-terminal fragment of the short nonmuscle α-tropomyosin has been determined at a resolution of 0.98 Å. Tropomyosin (TM) is an elongated two-chain protein that binds along actin filaments. Important binding sites are localized in the N-terminus of tropo­myosin. The structure of the N-terminus of the long muscle α-TM has been solved by both NMR and X-ray crystallography. Only the NMR structure of the N-­terminus of the short nonmuscle α-TM is available. Here, the crystal structure of the N-terminus of the short nonmuscle α-TM (αTm1bZip) at a resolution of 0.98 Å is reported, which was solved from crystals belonging to space group P 3 1 with unit-cell parameters a = b = 33.00, c = 52.03 Å, α = β = 90, γ = 120°. The first five N-­terminal residues are flexible and residues 6–35 form an α-helical coiled coil. The overall fold and the secondary structure of the crystal structure of αTM1bZip are highly similar to the NMR structure and the atomic coordinates of the corresponding C α atoms between the two structures superimpose with a root-mean-square deviation of 0.60 Å. The crystal structure validates the NMR structure, with the positions of the side chains being determined precisely in our structure

Structural anatomy of Protein Kinase C C1 domain interactions with diacylglycerol and other agonists

Diacylglycerol (DAG) is a versatile lipid whose 1,2-sn-stereoisomer serves both as second messenger in signal transduction pathways that control vital cellular processes, and as metabolic precursor for downstream signaling lipids such as phosphatidic acid. Effector proteins translocate to available DAG pools in the membranes by using conserved homology 1 (C1) domains as DAG-sensing modules. Yet, how C1 domains recognize and capture DAG in the complex environment of a biological membrane has remained unresolved for the 40 years since the discovery of Protein Kinase C (PKC) as the first member of the DAG effector cohort. Herein, we report the high-resolution crystal structures of a C1 domain (C1B from PKC delta) complexed to DAG and to each of four potent PKC agonists that produce different biological readouts and that command intense therapeutic interest. This structural information details the mechanisms of stereospecific recognition of DAG by the C1 domains, the functional properties of the lipid-binding site, and the identities of the key residues required for the recognition and capture of DAG and exogenous agonists. Moreover, the structures of the five C1 domain complexes provide the high-resolution guides for the design of agents that modulate the activities of DAG effector proteins. Protein kinase Cs (PKCs) define a central DAG-sensing node in intracellular phosphoinositide signaling pathways that regulate cell growth, differentiation, apoptosis, and motility. The structures of PKC C1 domain complexes with DAG and 4 agonists reveal the molecular basis of ligand recognition and capture.N

Structure of a tropomyosin N-terminal fragment at 0.98 Å resolution

Tropomyosin (TM) is an elongated two-chain protein that binds along actin filaments. Important binding sites are localized in the N-terminus of tropo­myosin. The structure of the N-terminus of the long muscle α-TM has been solved by both NMR and X-ray crystallography. Only the NMR structure of the N-­terminus of the short nonmuscle α-TM is available. Here, the crystal structure of the N-terminus of the short nonmuscle α-TM (αTm1bZip) at a resolution of 0.98 Å is reported, which was solved from crystals belonging to space group P3(1) with unit-cell parameters a = b = 33.00, c = 52.03 Å, α = β = 90, γ = 120°. The first five N-­terminal residues are flexible and residues 6–35 form an α-helical coiled coil. The overall fold and the secondary structure of the crystal structure of αTM1bZip are highly similar to the NMR structure and the atomic coordinates of the corresponding C(α) atoms between the two structures superimpose with a root-mean-square deviation of 0.60 Å. The crystal structure validates the NMR structure, with the positions of the side chains being determined precisely in our structure

Data collection and refinement statistics.

a<p>Values in parentheses refer to the high-resolution shell.</p><p>Data collection and refinement statistics.</p

TnSeq of <i>Mycobacterium tuberculosis</i> clinical isolates reveals strain-specific antibiotic liabilities

<div><p>Once considered a phenotypically monomorphic bacterium, there is a growing body of work demonstrating heterogeneity among <i>Mycobacterium tuberculosis</i> (Mtb) strains in clinically relevant characteristics, including virulence and response to antibiotics. However, the genetic and molecular basis for most phenotypic differences among Mtb strains remains unknown. To investigate the basis of strain variation in Mtb, we performed genome-wide transposon mutagenesis coupled with next-generation sequencing (TnSeq) for a panel of Mtb clinical isolates and the reference strain H37Rv to compare genetic requirements for <i>in vitro</i> growth across these strains. We developed an analytic approach to identify quantitative differences in genetic requirements between these genetically diverse strains, which vary in genomic structure and gene content. Using this methodology, we found differences between strains in their requirements for genes involved in fundamental cellular processes, including redox homeostasis and central carbon metabolism. Among the genes with differential requirements were <i>katG</i>, which encodes the activator of the first-line antitubercular agent isoniazid, and <i>glcB</i>, which encodes malate synthase, the target of a novel small-molecule inhibitor. Differences among strains in their requirement for <i>katG</i> and <i>glcB</i> predicted differences in their response to these antimicrobial agents. Importantly, these strain-specific differences in antibiotic response could not be predicted by genetic variants identified through whole genome sequencing or by gene expression analysis. Our results provide novel insight into the basis of variation among Mtb strains and demonstrate that TnSeq is a scalable method to predict clinically important phenotypic differences among Mtb strains.</p></div

Bedaquiline reprograms central metabolism to reveal glycolytic vulnerability in Mycobacterium tuberculosis

The approval of bedaquiline (BDQ) for the treatment of tuberculosis has generated substantial interest in inhibiting energy metabolism as a therapeutic paradigm. However, it is not known precisely how BDQ triggers cell death in Mycobacterium tuberculosis (Mtb). Using C isotopomer analysis, we show that BDQ-treated Mtb redirects central carbon metabolism to induce a metabolically vulnerable state susceptible to genetic disruption of glycolysis and gluconeogenesis. Metabolic flux profiles indicate that BDQ-treated Mtb is dependent on glycolysis for ATP production, operates a bifurcated TCA cycle by increasing flux through the glyoxylate shunt, and requires enzymes of the anaplerotic node and methylcitrate cycle. Targeting oxidative phosphorylation (OXPHOS) with BDQ and simultaneously inhibiting substrate level phosphorylation via genetic disruption of glycolysis leads to rapid sterilization. Our findings provide insight into the metabolic mechanism of BDQ-induced cell death and establish a paradigm for the development of combination therapies that target OXPHOS and glycolysis

Data collection and refinement statistics.

a<p>Values in parentheses refer to the high-resolution shell.</p><p>Data collection and refinement statistics.</p

Activity and phylogeny of Rv2911 and Rv3330.

<p>A. The beta-lactam, meropenem, contains a D-Ala-like group (red) and irreversibly acylates penicillin-binding proteins, including Rv2911 and Rv3330. B. Enzyme acylation by peptidoglycan stem peptide, the first step in peptidoglycan carboxy- and trans-peptidation, highlighting the similarity of the terminal D-Ala leaving group (red) and the D-Ala-like acceptor moiety of DAP (purple). C. Neighbor-joining tree of <i>Peptidase_S11</i> family proteins of Actinobacteria fall into several clades. Of the two mycobacterial clades, Clade 1 (orange) contains Rv2911, and Clade 2 (blue) contains Rv3330. <i>E. coli</i> and <i>B. subtilis</i> Peptidase_S11 enzymes (green) form an out-group with several actinobacterial sequences. D. Maximum-likelihood tree of <i>Peptidase_S11</i> proteins from select mycobacterial species, <i>Corynebacterium diphtheriae</i>, <i>E. coli</i>, <i>and B. subtilis</i>. High bootstrapping values confirm the separation of mycobacterial sequences into two distinct clades.</p